Transport cycle of the ABC transporter ABCB1

Protein motion lies at the heart of function of molecular machines including membrane transporters. Structural and dynamic details of the catalytic cycle of the human ABC transporter ABCB1, a multispecific drug efflux pump involved in drug resistance and pharmacokinetics will be the focus of the proposed project. Computational biology has proven an invaluable tool to translate static crystallographic information into protein motion and to integrate structural and biochemical evidence into a synthetic view of the dynamics at the molecular level. Crystal structures of three ABC exporters (Sav1866, MsbA and mouse ABCB1) have been solved and the observed conformations allow for perceiving the conformational changes. The project aims at structurally characterizing the different state in the transport cycle by using a combination of computational modeling and experiments. The principle goal of the project is the identification of the access paths of substrates, the understanding of the broadly set, but clearly preferential specificity for amphiphilic cationic drugs, characterization of the transporter`s conformational response to substrate and ATP binding as well as ATP hydrolysis and of the cross-talk between the transmembrane domains and the nucleotide binding domains that lies at the heart of substrate transport. Results from this integrated approach, which combines extensive computer simulations with experiments are expected to allow for characterization of conformations the dynamics underlying the transport cycle and for improving our understanding of substrate recognition.

The human genome contains 44 ABC exporters, which all have two transmembrane domains that bind and transport substrate and two nucleotide binding domains, which bind and hydrolyse ATP. In this grant project we focus on the multidrug resistance transporter P-glycoprotein (ABCB1) and aim to structurally characterizing different states of the transport cycle by using a combination of computational modeling and experiments. We identified and characterized the binding site for propafenone drugs and derivatives thereof within the large substrate binding site. It is well established that ATP binding and its subsequent hydrolysis drives the transport process of ABC transporters. We discovered that ATP binding depends on the nature of the residues within the ATP binding site. The strength of ATP binding is smaller in a degenerate nucleotide binding site and the geometry is different. Many structural details of the conformational changes associated with the transport cycle remained disputed, despite the availability of several crystal structures from the ABCB subfamily. We found that ABCB1 assumes in the plasma membrane a different conformation as suggested by most crystal structures. The transmembrane domains do not separate or only by very small extend, because we could established that ABCB1 remains functional, if the extracellular domains are chemically linked to each other. ABCB1 is expressed at the blood-brain-barrier, in the intestine, kidney, liver and in macrophages and transports an extraordinarily diverse range of structurally unrelated drugs, xenobiotics and endogenous substrates. Cancer cells acquire resistance to chemotherapy when expressing ABCB1. Several of the ABC transporters are causally related to disease. Pharmacological modulation of ABC transporter function can be a valid therapeutic principle. It can be advantageous to inhibit ABC transporters in cancer therapy. Knowledge of the physiological relevant conformations is a requirement for rational drug design for inhibitors of the multidrug resistance transporters. Mutations of ABC transporters are associated with disease like gout (ABCG2), cholestasis (ABCB11) or cystic fibrosis (ABCC7). The results of this research project have uncovered new insights of ABCB1 transporter structure in the membrane environment. Structural knowledge is necessary for applying the concept of pharmacochaperoning in rational drug design, in which drugs are tailored to stabilize proteins. Mutated transporter can be stabilized during folding, rescued from degradation and their surface expression enhanced, thereby regaining function.